Challenge Area: 06 Enabling Technologies. Specific Challenge Topic: 06-GM-101 Structural Analysis of Macromolecular Complexes. A major challenge in chromatin biology and molecular cytology is how to study the macromolecular structures of specific epigenetic chromatin modifications in single cells at nanoscale resolution. The many post-translational modifications of histone proteins play critical roles in defining the biological functions of chromosomes. There are different sets of modifications associated with transcriptionally active chromatin, with inactive chromatin, with replicating chromatin, with sites of chromosome damage, and with key subnuclear compartments such as centromeres and telomeres. Defects in these epigenetic marks, in the enzymes and proteins that "read", "write", and "erase" them, have been found to occur in many human diseases, including cancer and neural degenerative syndromes. Furthermore, these epigenetic marks are key determinants in stem cell biology, and are important both in maintaining the pluripotent state and in driving differentiation. At present, there are no technologies that can visualize the macromolecular structures of epigenetic histone modifications in single cells at resolutions any better than approximately 200-300 nm. The resolution of light microscopy is limited by diffraction, and the resolution of electron microscopy is limited by lack of contrast. Biochemical techniques including mass spectroscopy, chromatin immunoprecipitation, and chromosome conformation capture, are making great strides in defining the functional combinations of histone marks, but they cannot image those structures within the nucleus or follow their dynamics in live cells. That is the challenge. To meet it, we have designed novel probes of histone modification by fusing multivalent binding domains to photoactivatable fluorophores and expressing these "decoder" constructs in cells. Using recent advances in super-resolution microscopy, the positions of these reporters can be determined with a localization precision of 6-10 nm, reconstructing the image of modifications at a resolution below the width of the 30 nm chromatin fiber. The goal of this project is to exploit this proof of principle and develop the technology to enable researchers to explore the macromolecular structures of chromatin at levels that are an order of magnitude more precise than is currently possible. Over the next two years we will address three major aims. (1) We will construct a high quality, versatile set of decoder constructs that represent all of the known histone modification binding motifs. (2) We will characterize the properties and binding specificities of these decoders by comparing their co-localization with traditional antibody probes, by conducting genome-wide sequencing of bound DNA in chromatin immunoprecipitations, and by imaging the reporters in three-dimensions and in live cells. (3) We will construct decoders that are predicted to have novel new binding specificities through the rational design of chimeric, artificial multivalent, and synthetic binding motif combinations. The results of these efforts will develop the technology to enable the routine visualization of the nanoscape of chromatin epigenetics. This will impact basic research by providing the tools, reagents, and protocols that will marshal a paradigm shift in how chromatin is studied. Moreover, since chromatin modifications have real practical importance for cancer, neural degeneracies, embryonic development, assisted reproductive services, and future stem cell therapies, the ability to image epigenetic marks rapidly, in single live cells, at nanoscale resolutions has the potential to radically improve the diagnosis, classification, and treatment modalities associated with a wide spectrum of human health issues. Complex modifications of proteins on the chromosomes have major roles in regulating cellular physiology and keeping cells normal and healthy. A severe limitation in studying how these modifications work is the fact that we cannot see them, observe their structural organizations, or watch how they come and go. Overcoming this limitation is a formidable challenge that will have enormous impact on both basic and clinical health science, including cancer, neurodegenerative syndromes, and stem cell therapies. This project will exploit breakthroughs in fluorescent light microscopy, and recent insights into the biophysics of the modifications, to develop technology that will enable researchers to see the structures of these modifications for the first time at nanoscale resolution in single live cells.